A Novel Function of Lactate Transporter MCT1 inGastric Restitution

Lisa Marrone

under the direction ofDr. Susan J. Hagen

Beth Israel Deaconess Medical Center

Research Science Institute

July 31, 2007

Abstract

We investigated the function of the monocarboxylate transporter MCT1 in restitution

of gastric surface mucous cells. Restitution is the repair mechanism by which cells migrate

to cover an injured area. Cell migration after injury is dependent upon glycolysis, which

under some conditions yields the waste product lactate. We used the bioflavenoid phloretin,

a known inhibitor of MCT1, to block lactate transport in migrating cells and determine the

overall effect of MCT1 on restitution. We found that inhibition of MCT1 with phloretin

significantly and dose-dependently inhibited restitution. Furthermore, under HCO3−-free

conditions that simultaneously inhibit the bicarbonate transporter NBC, there was a com-

plete inhibition of restitution after injury with no effect on viability. These results suggest

that MCT1 functions along with NBC to regulate intracellular pH and provide a mechanism

for lactate efflux during glycolysis. Thus, this newly discovered role of MCT1 in gastric

restitution could lead to novel strategies for both facilitating restitution in gastrointestinal

cells after injury and inhibiting metastasis of cancer and other cells that utilize glycolysis for

migration.

1 Introduction

The layer of epithelial cells that lines the inner surface of the body’s digestive system forms

a barrier between digestive fluids and surrounding tissues and organs in the body cavity [1].

In the stomach, the integrity of this layer is necessary to prevent acidic gastric fluids from

permeating into surrounding tissues, causing inflammation and widespread tissue damage.

Because it is particularly prone to injury from food and/or ingested drugs [2], the body has

evolved a repair mechanism called restitution that restores the structure and function of

injured stomach lining [3].

Surface mucous cells comprise most of the epithelium that lines the stomach lumen. The

mucus secreted by these cells protects the stomach from digested substances as well as from

its own secreted hydrochloric acid (HCl). Other protective mechanisms exist, such as the

secretion of bicarbonate, which neutralizes the acid, and the formation of tight junctions

between cells, which form the basis of the epithelial barrier [2]. Surface cells are arranged at

the top and upper sides of tubular gastric glands that extend downwards from the surface

towards underlying muscle layers (Figure 1) [4]. Injury occurs in those cells that line the

stomach lumen, where surface cells are most exposed [1]. Although all surface cells prior to

injury are columnar in shape, in the first step of restitution, uninjured cells from the gastric

pit transform into flat cells. These flattened cells are then able to migrate across the apical

mucosal surface to cover the wounded area. The new cells at the surface repolarize to form a

confluent layer by reestablishing the tight junctions that hold cells together in a tissue layer.

Restitution is complete when the flattened cells have completely polarized into columnar

epithelial cells. These newly populated cells are identical in structure and function to those

that were lost from the apical surface upon injury [3]. Most importantly, this repair is rapid,

taking place in two hours or less [2].

In the presence of oxygen, cells create energy in the form of ATP through a pathway

1

Figure 1: Left: Morphology of the gastric pit and gastric gland. After injury at the surface,cells at the bottom of the gastric pit migrate upwards to replenish denuded areas. Right:gastric pits open into the lumen of the stomach, while gastric glands extend downwardstowards underlying smooth muscle.

beginning in the cytoplasm and ending in the mitochondrion. In the cytoplasm, glucose is

broken down through a process called glycolysis, which yields two net ATP molecules. Upon

completion of glycolysis, glucose has been converted into a molecule called pyruvate. Al-

though glycolysis can occur under either aerobic or anaerobic conditions, cellular respiration

in mitochondria will only take place if oxygen is present. Under low O2 conditions, anaerobic

metabolism of pyruvate takes place, where pyruvate is reduced to lactate. [5]

Although it was initially thought that restitution would be dependent on energy from

mitochondrial respiration, recent studies showed that, in fact, glycolysis drives restitution.

Cell migration after injury is dependent on glycolysis, while the reformation of tight junctions

and cell polarization after migration is dependent on both glycolysis and respiration. Thus,

restitution can occur even under anaerobic conditions. Interestingly, cell migration in the

absence of oxygen occurs in many different cells, including tumor cells. [1]

Although glycolysis provides the energy needed for restitution, acidification due to the

2

Figure 2: Gastric surface cells contain four known ion channels. Of these four, the Na+/H+

exchanger and the Na+/HCO3− cotransporter are thought to play a role in pH regulation.

waste product lactate can poison migrating cells [3]. When intracellular pH becomes too low,

glycolysis is inhibited, cells are unable to migrate, and they quickly undergo programmed cell

death [6]. Thus, combined with an already high hydrogen ion (H+) concentration in the stom-

ach lumen, the presence of lactate inside the cell makes it imperative that ion transporters

are present to regulate intracellular pH and to reduce intracellular lactate concentrations [7].

It is believed that surface cells have ion transporters to regulate intracellular pH by rapidly

transporting H+ out of the cell or by rapidly transporting bicarbonate (HCO3−) into the cell

[3]. Currently, four distinct ion transporters have been identified within the gastric surface

cell: Na+/H+ exchangers (NHE), Na+/K+ ATPases, Na+/HCO3− cotransporters (NBC),

and Na+-K+-Cl− contransporters (Figure 2) [4]. Of these transport mechanisms, NHE and

NBC especially are thought to play a role in maintaining intracellular pH [3]. However,

recent studies demonstrated that other pH regulatory mechanisms must be activated during

restitution, because blockade of existing transporters had little effect on restitution. A

study by Hagen et. al. [3] showed that the compound DIDS, a potent inhibitor of NBC,

completely inhibits restitution. Contradictory to this result, cells incubated in HCO3−-free

3

conditions, which would also inhibit NBC, successfully undergo restitution, although slightly

less effectively than under standard conditions. This suggests the presence of another ion

transport mechanism that is inhibited by DIDS but not dependent upon HCO3−.

With high intracellular lactate concentrations predicted during restitution, we hypothe-

sized that a lactate transporter must be required for lactate efflux during restitution. Lactate

cannot freely cross a cell’s plasma membrane, but instead must be transported through a

special channel called a monocarboxylate transporter (MCT), which interestingly has H+-

coupled transport properties and thus also regulates intracellular pH [6]. The first MCT

cloned, MCT1, was shown to be present in surface mucous cells at the basolateral membrane

and is potently inhibited by bulky aromatic compounds such as the bioflavenoid phloretin

[7]. To date, however, no function for this MCT has been identified in surface mucous cells.

In this study, we set out to discover the first known role of MCT1 in surface mucous

cells. To do this, we inhibited MCT1 with phloretin, a known inhibitor of MCT1 [7], and

showed a significant reduction in restitution under standard conditions with no reduction

in cell viability. To test the hypothesis that DIDS blocks both NBC and MCT1 to inhibit

restitution, we also used phloretin in HCO3−-free conditions. This particular experiment

showed complete inhibition of restitution demonstrating the dependence of restitution on

both basolateral ion transport systems.

2 Materials and Methods

2.1 Preparation of RGM1 Cell Cultures

The cells used in this experiment were rat gastric mucosal (RGM1) cells, which are non-

transformed and immortalized surface cells. RGM1 cells were established by Dr. H. Matsui

of the Institute of Physical and Chemical Science (RIKEN) Cell Bank, Tsukuba, Japan [8].

Cells were grown in six-well plates and cultured in DMEM-F12 (1:1) supplemented with

4

10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin, and 0.25 µg/ml

amphotericin B [9]. All cells were starved in serum-free medium (DMEM-F12 containing

15 mM HEPES at pH 7.4) for 24 hours prior to experimentation. Cells were incubated at

37◦C under 5% CO2 in air. Experiments were performed in either standard (STD) buffer or

bicarbonate-free (BF) buffer at pH 7.4.

2.2 Preparation of Buffer Solutions

The experimental medium was STD buffer [Appendix A]. To determine the role of HCO3−

in the recovery of wounds, mannitol and HEPES were substituted for HCO3− to create a

bicarbonate-free (BF) buffer [Appendix A]. STD buffer contained (in mM): 147 Na+, 5 K+,

131 Cl−, 1.3 Mg2+, 1.3 SO42−, 2 Ca2+, 15 HEPES, 20 D-Glucose, and 25 HCO3

−. Total

osmolarity of STD buffer was 347.6 mOsm. BF buffer contained (in mM): 147 Na+, 5 K+,

131 Cl−, 1.3 Mg2+, 1.3 SO42−, 2 Ca2+, 25 HEPES, 20 D-Glucose, and 15 Mannitol. Total

osmolarity of BF buffer was 347.6 mOsm.

2.3 Preparation of Phloretin Solutions

In order to investigate the role of monocarboxylate transporters (MCT) in wound repair,

wounded gastric cells were treated with phloretin, a known inhibitor of MCT1. Phloretin

solutions were prepared in 100% DMSO and then diluted to 0 µM, 30 µM, and 60 µM in

STD or BF buffer. The final concentration of DMSO was 0.1%.

2.4 Wounding

In order to mimic gastric injuries that heal through the process of restitution, small circular

wounds were made in confluent monolayers of RGM1 cells. The wounds were made in

each well of the cell culture plates using a pencil-type mixer with Teflon tip. After gentle

5

wounding, cells were washed twice with 1.5 mL STD or BF buffer. Buffer was then replaced

with phloretin solutions. Two wells received 1.5 mL of 0 µM phloretin in STD of BF buffer,

two wells received 1.5 mL of 30 µM phloretin in STD of BF buffer, and two wells received 1.5

mL of 60 µM phloretin in STD of BF buffer. Following wounding, cell plates were imaged

and then returned to 37◦C.

2.5 Microscopy

Wounds were imaged 0, 4, and 8 hours after the addition of phloretin. Cell plates were

placed in the incubator chamber of a Nikon TE300 Inverted Microscope and photos were

taken using a Hamamatsu Orcha digital camera. Wound area was measured using IPLab,

purchased from Scanalytics, Inc.

2.6 Crystal Violet Viability Assay

Following the 8 hour experiment, a viability assay was conducted to ensure that the experi-

mental conditions did not effect cell viability. Culture medium from each well was aspirated

into a separate conical tube and reserved for pH measurements using a pH meter. All cells

were washed twice with 1.5 mL PBS. Cells were fixed with 100% ice cold methanol and in-

cubated for 15 min. at room temperature. Cells were stained with crystal violet (1% crystal

violet, 9% methanol in PBS). The stain was incubated for 5 min. at room temperature before

cells were washed 10 times with warm water. Cells were air dried overnight. Each well was

solubilized with 1.5 mL 0.5% SDS for 30 min at room temperature using an environmental

shaker. 50 µL of lysate from each well of the six-well plates was then transferred to a well of

a 96-well plate. 200 µL of 0.5% SDS was added to each well of the 96-well plate. Absorbance

was read at an excitation of 590 nm and emission of 640 nm. Data were calculated as percent

viability when compared to cultures that received no treatment.

6

2.7 Statistical Analysis of Results

Statistical analysis was done with SigmaStat software (Jandel Scientific Software, San Rafael,

CA). Data from wound area experiments (n=6/treatment) and viability experiments (n=6/

treatment) were averaged and reported as means ± standard error. A one-way ANOVA

test was performed to compare controls to each level of drug concentration, with differences

regarded as statistically significant at P<0.05. A t-test was performed to compare STD and

BF conditions, with differences regarded as statistically significant at P<0.05. For all data

presented, statistical differences were highly significant at P<0.001.

3 Results

3.1 Restitution in Control RGM1 Cells

Control cells were grown in STD buffer and thus contained functioning NBC and MCT

transporters. After wounding and incubation for 4 hours in STD buffer, wound area was

(85.1 ± .7)% of initial wound area. By 8 hours, control wound area was (73.2 ± .6)% of

initial wound area (Figure 3A and C).

3.2 Restitution in HCO3−-free Conditions

HCO3−-free (BF) conditions were used to test the hypothesis that blockade of NBC inhibits

restitution in RGM1 cells. After a 4 hour incubation, the wounds of cells grown in BF

buffer in the absence of phloretin had restituted to (88.8 ± .5)% of initial wound area. By

eight hours, BF wounds had restituted to (80 ± 1)% of initial wound area (Figure 3A

and C). That cultures incubated in BF conditions restituted at a rate significantly slower

than that of cultures in STD buffer demonstrates that the presence of a HCO3− transport

mechanism, such as NBC, is required for restitution in RGM1 cells (t=-6.031, P<0.001).

7

Figure 3: A: Images of wounded RGM-1 cells under both STD and BF conditions at time0 hours and 8 hours. B: Images of wounded RGM-1 cells incubated with 60µM phloretinunder both STD and BF conditions at time 0 hours and 8 hours. C: A graph of the effectof phloretin solutions on restitution in cultured cells (n=6/treatment, P<0.001).

8

Figure 4: Cell viability data in STD and BF buffer with varying concentrations of phloretin.It was determined that neither phloretin nor HCO3

−-free conditions compromised cell via-bility (n=6/treatment, p<0.05).

However, considering the lack of the full inhibition of restitution that occurs when DIDS is

added to inhibit NBC, other DIDS-inhibitible transporters, such as MCT1, must be required

for restitution.

3.3 Restitution in Phloretin Solutions

Phloretin was used in varying concentrations to test the effect of MCT1 inhibition on resti-

tution in cultured cells (Figure 3B). At a concentration of 30µM phloretin in STD buffer,

restitution was (92.1 ± .3)% at 4 hours and (84.9 ± .5)% at 8 hours. At a concentration of

60µM phloretin, restitution was (94.2 ± .3)% at 4 hours and (90.5 ± .5)% at 8 hours (Figure

3B and C). At a concentrations of 30µM phloretin in BF buffer, restitution was (95.3 ±.4)% at 4 hours and was (92.3 ± .5)% at 8 hours (Figure 3C). 60µM phloretin in BF buffer

completely inhibited restitution, with only (1.7 ± .4)% recovery by 8 hours (Figure 3B and

C). Results at each phloretin concentration were statistically significant from control levels

9

under both STD and BF conditions (P<0.001). That inhibition of MCT1 with phloretin

significantly and dose-dependently inhibits restitution shows that MCT1 in conjunction with

NBC is critical for maximum wound repair in RGM1 cells.

3.4 Cell Viability

A crystal violet viability assay was conducted in order to ensure that the experimental

conditions did not affect cell viability. It was found that cells were confluent and 100%

viable after 8 hours of incubation with all experimental solutions (Figure 4).

4 Discussion

In this study, bicarbonate-free conditions were used in conjunction with phloretin to test the

hypothesis that DIDS inhibits restitution by blocking both MCT1 and NBC, rather than

NBC alone. Using bicarbonate-free buffer to inhibit NBC and phloretin to inhibit MCT1, it

was found that although inhibition of NBC has an effect on restitution, the effect of MCT1

inhibition was three times greater. This result suggests that MCT1 activity is essential

for attaining the maximum rate of restitution. These results are novel and have not been

reported for other migration or wounding studies.

Although phloretin potently inhibits MCT1 at low concentrations [7], the drug is non-

specific and blocks other ion transporters, channels, and intracellular signaling pathways.

First among these are members of the protein kinase C (PKC) family [10], protein kinases

that have been implicated in a variety of cellular functions including proliferation, apoptosis,

differentiation, motility and inflammation [11]. PKC is found in a variety of cell types, in-

cluding the gastric epithelia [11]. Recent experiments have demonstrated that inhibition of

PKC inhibits restitution in guinea pig gastric mucosa [12], rabbit gastric epithelial cells [13],

and colonic epithelial cells [14]. However, inhibition of PKC has been demonstrated to have

10

an inhibitory effect on restitution only at concentrations of 200-300µM. In fact, phloretin is

most specific for inhibiting PKC at concentrations of 200-250µM [15], about 4-fold higher

than those used in this experiment. Although it is unlikely that our concentrations of 30 and

60 µM phloretin were sufficient to inhibit restitution via PKC inhibition, positive control

studies should be conducted using GF 109203X, another known PKC inhibitor [16].

Phloretin is also known to activate large conductance Ca2+-activated-K+ channels (BK(Ca))

[17]. However, although there is evidence that BK(Ca) channels are present in smooth muscle

[18] [19] and neurons [20], there is no literature to support the presence of BK(Ca) in gastric

epithelial cells. Positive control studies could be conducted with 1,3-dihydro-1-[2-hydroxy-5-

(trifluoromethyl)phenyl]-5-trifluoromethyl-2H-benzimidazol-2-one, another known activator

of BK(Ca) [21], to rule-out involvement of this channel in restitution.

Lastly, phloretin is a known inhibitor of many isoforms of glucose channels [22]. Although

the specific glucose channels present in the gastric mucosa are not known, a study in frog

gastric tissue conducted by Hagen et al. [1] showed that glucose-free conditions had no

inhibitory effect on restitution. Preliminary data in RGM1 cultured cells for this study also

indicate no inhibitory effects of glucose-free buffer on restitution.

Thus, given that the other known effects of phloretin are unlikely to account for our

results, we conclude that the inhibition we see in this experiment is due to phloretin inhibition

of MCT1.

The discovery that MCT1 is critically involved in restitution could support a novel model

for both the regulation of energy and pH regulation in migrating gastric surface cells after

injury. Although cell migration in restitution is driven by glycolysis, which under some con-

ditions yields the waste-product lactate, accumulation of lactate within the cell can actually

inhibit migration and lead to programmed cell death. As an H+-coupled lactate transporter,

MCT1 can transport lactate molecules through the otherwise impermeable cellular mem-

brane and additionally prevent intracellular acidification by the efflux of H+, allowing cell

11

migration to continue. We hypothesize that phloretin’s potent inhibitory effects on restitu-

tion are demonstrative of its interference with MCT’s pH regulation mechanism. However,

further studies will be needed to fully elucidate the effects of MCT1 on restitution by specific

blockade of MCT1 using siRNA technology.

Our results are immediately applicable to the study of maintenance of the gastric mucosa.

Rapid restitution is required both in daily life and in disease to prevent acidic gastric juices

from causing tissue damage and ulcers. We show that the rapid and efficient restitution that

drives both daily maintenance and repair after disease is dependent upon MCT1 activity.

In addition, the newly discovered role of MCT1 in migrating cells during gastric restitution

can potentially extend to existing models of migration in other cell types, especially those

that migrate under either aerobic or anaerobic conditions by utilizing glycolysis, such as the

repair of skin epithelial cells and the migration of lymphocytes and cancer cells. In this way,

our work in gastric restitution could lead to novel strategies for both facilitaing restitution

in skin cells after burn injury and inhibiting metastasis of cancer.

5 Conclusion

We have found that inhibition of MCT1 with phloretin and concurrent inhibition of NBC

with HCO3−-free conditions results in complete inhibition of restitution after injury. In

individual experiments, HCO3−-free conditions had only a minimal effect on restitution,

suggesting that bicarbonate transport by NBC is not particularly critical for restitution.

However, the significant inhibition of restitution effected by phloretin in standard physio-

logical conditions suggests that MCT1 is essential to attain maximum rates of restitution.

Although phloretin is nonspecific, inhibiting other ion transporters, channels, and intracellu-

lar signaling pathways, we eliminated such nonspecific effects as explanations for our results

through analysis of dosage thresholds and preliminary data on the inhibition of other ion

12

channels. Our results show that maintenance and repair after disease of the gastric mucosa

is dependent upon MCT1 activity. In addition, this novel role of MCT1 can possibly extend

models of migration in other cell types, especially those that require glycolysis to migrate

under aerobic or anaerobic conditions.

6 Acknowledgments

I would like to thank my mentor, Dr. Susan J. Hagen, at the Beth Israel Israel Deaconess

Medical Center for her continued support and mentorship. I thank Dr. Muvaffak and Boram

Cha for providing me with cell cultures. I greatly appreciate the time and effort taken by

my tutor Viviana Risca in providing suggestions on my paper.

13

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A Buffer Recipes

Compound mM FW 1L (g) 100mL (g) 500mL (g)NaCl 122 58.44 7.13 0.713 3.57KCl 5 74.56 0.373 0.0373 0.187MgSO4 1.3 246.5 0.320 0.032 0.16CaCl2 2 147.02 0.294 0.0294 0.147HEPES 15 238.31 3.58 0.358 1.79D-glucose 20 180.16 3.603 0.36 1.80NaHCO3

− 25 84.01 2.1 0.455 2.28

Table 1: Recipe for standard buffer

Compound mM FW 1L (g) 100mL (g) 500mL (g)NaCl 122 58.44 7.13 0.713 3.57KCl 5 74.56 0.373 0.0373 0.187MgSO4 1.3 246.5 0.320 0.032 0.16CaCl2 2 147.02 0.294 0.0294 0.147Na+-HEPES 25 260.3 6.51 0.651 3.255D-glucose 20 180.16 3.603 0.36 1.80Mannitol 15 182.2 2.73 0.273 1.37

Table 2: Recipe for bicarbonate-free buffer

17